Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

BackgroundProtein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease.ResultsHere we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs) in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen’s annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100–150 amino acids), with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300–400 amino acids), with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons.ConclusionsWe employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f.sp. hordei. Based on relative intron position and the distribution of CSEPs with a ribonuclease-like domain in the phylogenetic tree we hypothesize that the associated genes originated from an ancestral gene, encoding a secreted ribonuclease, duplicated successively by repetitive DNA-driven processes and diversified during the evolution of the grass and cereal powdery mildew lineage.

Patternrecognition receptors in eukaryotes initiate defence responses on detection of microbe-associated molecular patterns shared by many microbe species. The Leu-rich repeat receptor-like kinases FLS2 and EFR recognize the bacterial epitopes flg22 and elf18, derived from flagellin and elongation factor-Tu, respectively. We describe Arabidopsis 'priority in sweet life' (psl) mutants that show de-repressed anthocyanin accumulation in the presence of elf18. EFR accumulation and signalling, but not of FLS2, are impaired in psl1, psl2, and stt3a plants. PSL1 and PSL2, respectively, encode calreticulin3 (CRT3) and UDP-glucose:glycoprotein glycosyltransferase that act in concert with STT3A-containing oligosaccharyltransferase complex in an N-glycosylation pathway in the endoplasmic reticulum. However, EFR-signalling function is impaired in weak psl1 alleles despite its normal accumulation, thereby uncoupling EFR abundance control from quality control. Furthermore, salicylic acid-induced, but EFR-independent defence is weakened in psl2 and stt3a plants, indicating the existence of another client protein than EFR for this immune response. Our findings suggest a critical and selective function of N-glycosylation for different layers of plant immunity, likely through quality control of membrane-localized regulators.

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVRa gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVRa genes and identified AVRa1 and AVRa13, encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVRa1 and AVRa13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVRA1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVRA1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVRA1. Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

Nucleotide-binding domain and leucine-rich repeat (NLR)-containing proteins in plants and animals mediate intracellular pathogen sensing. Plant NLRs typically detect strain-specific pathogen effectors and trigger immune responses often linked to localized host cell death. The barley Mla disease resistance locus has undergone extensive functional diversification in the host population and encodes numerous allelic NLRs each detecting a matching isolate-specific avirulence effector (AVRA) of the fungal pathogen Blumeria graminis f. sp. hordei (Bgh). We report here the isolation of Bgh AVRa7, AVRa9, AVRa10, and AVRa22, which encode small secreted proteins recognized by allelic MLA7, MLA9, MLA10, and MLA22 receptors, respectively. These effectors are sequence-unrelated, except for allelic AVRa10 and AVRa22 that are co-maintained in pathogen populations in the form of a balanced polymorphism. Contrary to numerous examples of indirect recognition of bacterial effectors by plant NLRs, co-expression experiments with matching Mla-AVRa pairs indicate direct detection of the sequence-unrelated fungal effectors by MLA receptors.